Synthesis of β-lactam antibacterials using soluble side chain esters and enzyme acylase

ABSTRACT

Disclosed is a process for the synthesis of β-lactam antibacterials using soluble side chain esters in the presence of enzyme acylase. Also disclosed are novel esters useful as reactants in said process.

This application is a divisional application of Ser. No. 08/895640 filedJul. 17, 1997 now U.S. Pat. No. 5,922,907 claims priority fromprovisional U.S. application Ser. No. 60/022,622, filed Jul. 26, 1996,incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to a process for the production ofantibacterial β-lactams, namely penicillins and cephalosporins, in thepresence of the enzyme, acylase, and a soluble ester as the acyl source.The invention is also directed to novel esters useful as reactants insaid process.

DESCRIPTION OF RELATED ART

Japanese Patent Application Publication Nos. 47-25388, 47-29588,48-26985, 48-35090, 48-99393, 49-14687, 49-36890, 49-48892, 49-75787,49-134893 and 52-110896 teach processes for preparing penicillins orcephalosporins by reacting a methyl or ethyl ester of the acyl moiety tobe introduced, with 6-aminopenicillanic acid or 7-aminocephalosporanicacid or derivatives thereof. The acyl source in these references is alower alkyl ester and especially a methyl ester. Eiji Kondo, et al.,U.S. Pat. No. 4,340,672, issued Jul. 20, 1982, discloses a process forpreparing antibacterial β-lactams, that is penicillins andcephalosporins, by the action of an acylase on a penicillin orcephalosporin nucleus amine and an ester(1) of the following formula asthe acyl source ##STR1## wherein RCO is an acyl group of penicillin orcephalosporin side chains; X is a hydrogen atom, lower alkyl group orhydroxy lower alkyl group; Y is a hydrogen atom or a lower alkyl group;and n is an integer from 1 to 20. The foregoing process uses the(poly)ethyleneglycol ester as the acyl source which is freely misciblewith water as compared to known acyl sources, such as methyl esters,which have low solubility.

WO 92/01061, assigned to Novo Nordisk and published Jan. 23, 1992,relates to a process for the preparation of β-lactam derivatives byenzymatic acylation of the parent amino β-lactam with amides asacylating agents.

SUMMARY OF THE INVENTION

The present invention provides a novel process for preparingantibacterial β-lactams, that is penicillins and cephalosporins, by theaction of an acylase on a penicillin or cephalosporin nucleus amine andan ester having the formula (II) as the acyl source ##STR2## wherein RCOis a penicillin or cephalosporin side chain acyl group; R' is H or CH₂ Xor CHXY; R" is H or CH₂ Y or CHXY and X and Y each are independentlyhydrogen, hydroxy, lower alkyl or hydroxy-lower alkyl.

DETAILED DESCRIPTION OF THE INVENTION

As used in the specification, unless otherwise indicated explicitly orby context, the acyl group represented by RCO is an acyl of natural orsynthetic, penicillins or cephalosporins. The lower alkyl includes alkylof 1 to 4 carbon atoms.

One aspect of the present invention provides a process for theproduction of β-lactam antibacterials by reacting an ester of theformula (II) with aminoazetidinone carboxylic acid of the formula (III)in the presence of acylase in an aqueous medium to produce the β-lactamantibacterial of the formula (IV): ##STR3## wherein RCO is an acylgroup, NH₂ --Q is an amino source and acylase is an enzyme of bacterialor fungal origin or obtained by recombinant methods. The substituents R'and R" are as defined above. The acyl group RCO can be straight,branched, cyclic, or partially cyclic lower alkanoyl or lower alkenoyl;monocyclic lower aralkanoyl, monocyclic aryloxylower alkanoyl, (O, N, orS)-heterocyclic-lower alkanoyl, (O, N, or S)-heterocyclic thio-loweralkanoyl, cyanoacetyl, cyanomethyl-thioacetyl, monocyclic arylglycyl,monocyclic cycloalkenylglycyl, monocyclicarylglycolyl, N-acyl-ariglycyl,monocyclicarylmalonyl or arylsulfoalkanoyl, all above optionally havinglower alkyl, aminomethyl, halogen, hydroxy, lower alkanoyloxy or loweralkoxy as a substituent, and preferably containing 1 to 15 carbon atoms.

The preferred RCO groups are D-phenylglycyl and 4-hydroxy-D-phenylglycylwhich are well known as side chains of antibiotics such as cephalexinand amoxicillin.

The amino source NH₂ --Q refers to the substituent on the 7-amino groupor 6-amino group of known cephalosporin or penicillin antibiotics, Qhaving the formula (VI): ##STR4## wherein Z is hydrogen, halogen, loweralkyl of 1-3 carbon atoms, haloalkyl of 1-3 carbon atoms, C₂₄ alkenyl, anucleophilic group or lower alkyl of 1-3 carbon atoms substituted by anucleophilic group. The halogen atom is chlorine, bromine or iodine andthe representative nucleophilic groups are disclosed in Japanese PatentApplication Publication No. 49-81381.

The enzyme acylase can be of plant, animal, fungal or bacterial originor obtained by recombinant methods. The bacterial or fungal acylases areespecially important as far as production, efficiency, cost andstability are concerned.

Representative bacteria or fungi for the acylase source include strainsof microorganisms belonging to, for example, genera Acetobacter,Achromobacter, Aeromonas, Alkaligenes, Arthrobacter, Brevibacterium,Beneckea, Bacillus, Corynebacterium, Escherichia, Flavobacterium,Gluconobacter, Kluyvera, Microbacterium, Micrococcus, Nocardia, Proteus,Pseudomonas, Rhodopseudomonas, Spirillum, Staphylococcus, Xanthomonas,Aphanociadium or Cephalosporium, or natural or artificial mutants orvariants of them capable of producing acylase for the reaction of thisinvention. Such strains include those described in, for example,Advances in Applied Microbiology, Volume 20, page 217 (1976), or naturalor artificial mutants of them available for the reaction of thisinvention. An especially useful enzyme is penicillin G amidase which canbe obtained from Escherichia coli by methods which are well known in theart. It can also be obtained by use of recombinant E. coli containingthe penicillin G amidase gene with a tac promoter by methods which arewell known in the art. This method is preferred since it makes availablelarge quantities of the enzyme suitable for the manufacturing process.

The aminoazetidinone carboxylic acid of formula (III) can be used aswater soluble alkali metal salts, such as sodium, lithium and potassium,or as an ester, such as methanesulfonylethyl ester or acetonyl ester.The aminoazetidinones of formula (III) and its salts or esters are knowncompounds and can be prepared by known methods.

The esters of formula (II) can be prepared by known methods, that is, byreaction of an acid RCOOH with a diol or triol or polyol of the formula(V): ##STR5## wherein RCO, R' and R" are as defined above. Theesterificiation reaction can be by dehydration under acid catalyzedconditions or by means of acid halide or by ester exchange reactions.The RCOOH and the diol, triol or polyol are known compounds or can beobtained by methods which are well known in the art.

Another aspect of the invention provides the ester R--COO--CH₂ --CH₂--OH wherein R is D-phenylglycyl or 4-hydroxy-D-phenylglycyl. Theseesters are novel and important reactants in the process of the inventionbecause they provide yields of over 90% of the final products. Theseesters are prepared by known methods.

A preferred embodiment of the present invention is a process forpreparing antibacterial β-lactams, that is, cefprozil, cefadroxil andamoxicillin by the action of penicillin G amidase on penicillin orcephalosporin amines of the following formula (VII): ##STR6## wherein Zis methyl (in 7-ADCA) or I-propenyl (in 7-PACA, the cefprozil nucleus)group and an ester of the formula (VIII):

    R--COO--CH.sub.2 --CH.sub.2 --OH                           (VIII)

wherein R is 4-hydroxy-D-phenylglycyl. The penicillin or cephalosporinamines of the formula (VII) are known compounds and can be obtained bymethods well known in the art. The ester of formula (VIII) can beobtained by reacting RCOOH wherein R is defined as above with ethyleneglycol. The compound RCOOH and ethylene glycol are commerciallyavailable.

The process of the invention is carried out by preparing a solutioncontaining ester, cephalosporin or penicillin amine and the enzymewithout a buffer at room temperature. This solution is prepared bydissolving the ester in water and adding ammonium hydroxide until a pHof 7.5 is reached. Then the penicillin or cephalosporin amine is addedto the ester solution and the pH again adjusted to 7.5 with ammoniumhydroxide. The mixture is then cooled to 5-15° C. and the solid enzymeis added to it. During this time, the pH falls about 0.6 units and isnot maintained at 7.5. The reaction mixture is then analyzed by highpressure liquid chromatography, for example on a C18 reversed phasecolumn, 5 cm×4.6 mm, 5 μm spherosorb ODS2. The final product is obtainedin a yield of 90-99%.

The process of this invention gives the final antibacterial β-lactam inthe usual yield of over 90% and sometimes over 95% and occasionally over99%. The yield obtained by simple alkyl esters, such as methyl areusually in the range of 70 to 90%. Because of the high yield of thefinal antibiotic obtained by the present process, the process is veryuseful for the manufacture of penicillin or cephalosporin antibiotics.

A typical reaction mixture contains 5-35% of the acyl source (VIII)(preferably 8-12%) and 2-20% of the amine source (VII) (preferably2-5%). The enzyme can be present in a soluble or insoluble form, butpreferably the enzyme is present in the form of an insoluble immobilizedpreparation. This preparation has advantages, such as improved stabilityof the enzyme and also relative ease of removal of immobilized enzymefrom reaction mixtures as a first step in the isolation of theantibiotic.

The invention is illustrated but in no way limited by the followingExamples:

EXAMPLE 1

Synthesis of cefprozil from (VII) using immobilized recombinantpenicillin G amidase as the enzyme and hydroxyethyl ester of4-hydroxy-D-phenyiglycine prepared by acid catalyzed reaction as theacyl source

A mixture of 4-hydroxy-D-phenylglycine (10 g), ethylene glycol (15 ml)and concentrated sulphuric acid (5 ml) was stirred for 18 hours at 55°C. under anhydrous conditions. The solution was cooled, and then ice (10g) was added to it, and the pH was adjusted to 1.0 with 10 N--NH₄ OH(4.5 ml) giving 40 ml of solution of hydroxyethyl ester.

The enzyme mixture of 20 ml containing 10% ester, 4% (VII), and 8%enzyme (equivalent to 32 IU/ml of enzyme) was made up without buffer asfollows:

The above prepared ester solution (6.9 ml) was mixed with water (2 ml)and adjusted to pH 7.5 with 10 N--NH₄ OH. Then the compound (VII) (0.8g) was added to it and the pH adjusted to 7.5 with 1 N-NH₄ 0H and thevolume to 18.4 ml. Then the mixture was cooled to 5-15° C. and solidenzyme (1.6 g; 640 IU) was added to it. The pH was not maintained at 7.5and fell about 0.6 units during the reaction. The reaction mixture wasanalyzed by HPLC on a C18 Reverse Phase column 5 cm×4.6 mm, 5 μmspherosorb ODS2. The mobile phase was 10% acetonitrile/0.3% H₃ PO₄ ©2mil/minute with 215 nm detection. The isomers of cefprozil appeared at2.9 minutes (cis) and at 5.1 minutes (trans). The final product wasobtained with a maximum yield of 92-96%. The whole experiment wascompleted in 25-50 minutes.

EXAMPLE 2

Synthesis of cefprozil from (VII) using immobilized recombinantpenicillin G amidase as the enzyme and purified hydroxyethyl ester of4-hydroxy-D-phenylglycine as the acyl source

The purified ester was prepared by the reaction of the Dane salt of4-hydroxy-D-phenylglycine with bromoethanol and was obtained as a purecrystalline hydrochloride salt.

The reaction mixture of 20 ml containing 12.5% ester, 4% (VII), and 8%enzyme (equivalent to 24 IU/ml of enzyme) was prepared as follows:

The purified ester (2.5 g), prepared as above, was mixed with water (9ml) and adjusted to pH 8.0 with concentrated NH₄ OH to give a solution.The compound (VII) (0.8 g) was added to the solution and the pH of thesolution adjusted to 6.76 with 2M--H₂ SO₄ and the volume to 18.4 ml.This solution was cooled to 15° C. and then solid enzyme (1.6 g; 480 IU)was added to it. The pH was not maintained and fell about 0.6 unitsduring the reaction. The final product was obtained in a maximum yieldof 99%. The area percent of unreacted (VII) fell to 0.7%. The experimentwas completed in twenty-five minutes.

EXAMPLE 3

Synthesis of cefadroxil from 7-ADCA using Boehringer penicillin Gamidase as the enzyme and hydroxyethyl ester of4-hydroxy-D-phenylglycine prepared by acid catalyzed reaction as theacyl source

The ester was prepared as in Example 1. The experiment was repeated asin Example 1 but at 15° C. and using 1.6 g of the Boehringer enzyme. Thereaction mixture was analyzed by HPLC on a Merck column, 250-4,LiChrosorb RP-18 (10 μm), with 10% acetonitrile/1% NH₄ H₂ PO₄ as mobilephase run at 1.3 ml/minute with 280 nm detection. The product wasobtained with a maximum yield of ˜95%. The experiment was completed in35 minutes.

EXAMPLE 4

Synthesis of amoxicillin from 6-APA using Boehringer penicillin Gamidase as the enzyme and hydroxyethyl ester of4-hydroxy-D-phenylglycine prepared by acid catalyzed reaction as theacyl source

A mixture of volume 20 ml containing 12% ester, 3% 6-APA, and 8% enzymewas prepared as follows:

A solution of the ester (prepared as in Example 1, 8.3 ml) was mixedwith water (2 ml) and adjusted to pH 7.5 with 10 N--NH₄ OH. Then thecompound 6-APA (0.6 g) was added to the solution and the pH adjusted to˜7.0 with 1 N ammonium hydroxide to a volume to 18.4 ml. Then thesolution was cooled to 20° C. and Boehringer enzyme (1.6 g) was added toit. This solution was analyzed by HPLC on a C18 Reverse Phase column 5cm×4.6 mm, 5 μm spherosorb ODS 2. The mobile phase was 2.5%acetonitrile/0.025M NaH₂ PO₄ to pH 3.5 with H₃ PO₄ run@1 mI/minute with215 nm detection. The final product was obtained in a yield of 90-95% in160 minutes.

What is claimed is:
 1. A process for producing antibiotic cefprozil,cefadroxil or amoxicillin, comprising reacting: ##STR7## wherein Z ismethyl or 1-propenyl group with an ester of the formula

    R--COO--CH.sub.2 --CH.sub.2 --OH

wherein R is 4-hydroxy-D-phenylglycyl in the presence of the enzyme,penicillin G amidase to produce cefprozil, cefadroxii or amoxicillin. 2.The process of claim 1, wherein the enzyme is immobilized recombinantpenicillin G amidase.